Dog Rose (Rosa canina L.) is a wild native species in Iran, with a significant genetic diversity. The purpose of this research was to study quantitative and qualitative characteristics of saturated and unsaturated fatty acids dog rose ecotypes in five habitats of Isfahan province in 2014-2015. Oil was extracted from seeds by using soxhlet apparatus; the fatty acid composition of oil was characterized and quantified using gas chromatography (GC) in the laboratory of Islamic Azad University, Isfahan (Khorasgan) Branch. According to the results, the highest and the lowest oil percentage were observed in ecotypes from Miyankish-kashan (11.43%) and Aghcheh-Buin va Miyandasht (7.89%), respectively. The highest amount of unsaturated acids including linoleic, linolenic, oleic, palmitoleic and arachidonic acid were obtained in Kopehjamshid-Tiran (55.00%), Zarneh-Buin va Miyandasht (28.38%), Sadeghiyeh-Fereydonshahr (22.58%), Aghcheh-Buin va Miyandasht (0.22%) and Zarneh-Buin va Miyandasht (0.12%), respectively. Sadeghiyeh-Fereydonshahr had the lowest amounts of saturated acids of palmitic (3.69%) and stearic acids (1.77%), respectively. The ecotypes from Kopehjamshid-Tiran, Zarneh-Buin va Miyandasht, and Sadeghiyeh-Fereydonshahr had the lowest amount of myristic acid. Seed fatty acid analysis showed the minimum amount of total unsaturated fatty acids in Sadeghiyeh- Fereydonshahr (93.87%) and its minimum in Aghcheh-Buin va Miyandasht (91.51%), respectively. In this research, seed oil of Dog rose ecotypes from the studied habitats have unique nutritional characteristics and medicinal properties due to a high percentage of unsaturated fatty acids and an appropriate omega-6 to omega-3 ratio.